Plant Cell Physiol. 44(10): 1027–1036 (2003)
JSPP © 2003
Ca2+-Dependent Cessation of Cytoplasmic Streaming Induced by Hypertonic
Treatment in Vallisneria Mesophyll Cells: Possible Role of Cell Wall–Plasma
Membrane Adhesion
Teruyuki Hayashi 1, 2 and Shingo Takagi
Department of Biology, Graduate School of Science, Osaka University, 1-16 Machikaneyama-cho, Toyonaka, Osaka, 560-0043 Japan
;
In mesophyll cells of the aquatic angiosperm Vallisneria gigantea Graebner, a rapid and transient inhibition of
cytoplasmic streaming was induced by hypertonic treatment with sorbitol. Higher concentrations of sorbitol
induced the response more rapidly and in more cells. The
response to hypertonic treatment was strictly dependent on
the presence of extracellular Ca2+ and was sensitive to Ca2+channel blockers, including the stretch-activated Ca2+channel blocker Gd3+. Deplasmolyzed cells never responded
to a second hypertonic treatment administered immediately after plasmolysis and subsequent deplasmolysis.
Responsiveness was gradually recovered during 24 h of
incubation; however, cycloheximide, cordycepin, and trypsin
completely suppressed the recovery. Although an Arg-GlyAsp (RGD) hexapeptide markedly disturbed the pattern of
cytoplasmic streaming, it exhibited no specific effects on the
response to hypertonic treatment or on the recovery of
responsiveness. Taken together, our results demonstrate that
leaf mesophyll cells in a multicellular plant can respond to
mechanical stimuli and that a Ca2+ influx through stretchactivated Ca2+ channels plays an indispensable role in the
response. Furthermore, the possible involvement of RGDinsensitive but trypsin-sensitive protein factor(s), whose function is impaired by detachment of the plasma membrane
from the cell wall, is suggested.
Keywords: Cell wall–plasma membrane adhesion — Cytoplasmic streaming — Hypertonic treatment — Mechano-perception
— Stretch-activated Ca2+ channel — Vallisneria gigantea
(mesophyll cells).
Abbreviations: APW, artificial pond water; [Ca2+]cyt, cytoplasmic
concentration of Ca2+; EGTA, ethylene glycol bis(b-aminoethylether)N,N,N¢,N¢-tetraacetic acid; PIPES, piperazine-1,4-bis(2-ethanesulfonic
acid).
Introduction
Plant cells respond to various kinds of mechanical stimuli, including touch (Knight et al. 1991, Staves and Wayne
1
2
1993, Shimmen 1996, Legué et al. 1997), wind (Knight et al.
1992), gravity (Wayne et al. 1992, Legué et al. 1997), and
extracellular changes in osmotic pressure (Okazaki and Tazawa
1986, Tazawa et al. 1995, Knight et al. 1997, Takahashi et al.
1997). Mechanical stimuli induce a variety of cellular responses,
and Ca2+ has been demonstrated to be involved in many of
those responses, for example, touch-induced rapid turgor
movement in Mimosa pudica (Campbell and Thompson 1977)
and thigmotropism of Zea mays roots (Millet and Pickard
1988). Since a number of studies have revealed that the cytoplasmic concentration of Ca2+ ([Ca2+]cyt) rapidly and transiently increases after perception of mechanical stimuli (Knight
2000, and references therein), Ca2+ has been postulated to play
the essential role of second messenger at the diverging point in
signaling processes. Mechanical stimuli produce deformation
in the plasma membrane, and it is thought that such deformation activates mechano-sensitive ion channels in both animal
(Yang and Sachs 1989) and plant cells (Pickard and Ding
1993). The presence of Ca2+-permeable ion channels activated
by stretching of the plasma membrane has been suggested in
plant cells such as guard cells of Vicia faba (Cosgrove and
Hedrich 1991), epidermal cells of sweet red onion (Ding and
Pickard 1993), and tobacco protoplasts (Falke et al. 1988).
Recently, a gene encoding a stretch-activated Ca2+-permeable
ion channel (Mid 1) has been identified in yeast (Kanzaki et al.
1999). Such Ca2+-channel activities may play a central role in
the increase in [Ca2+]cyt induced by mechanical stimuli.
Compared with the extensive characterizations of downstream responses induced by mechanical stimuli (Knight 2000,
and references therein), much less is known about the mechanisms for mechano-perception in plant cells. In animal cells,
focal adhesions play a central role in sensing physical forces
and transducing mechano-sensory signals into the cells (Ingber
1991). At focal adhesions, extracellular matrix proteins such as
vitronectin and fibronectin, which are equipped with an RGD
(Arg-Gly-Asp) motif, bind using the RGD motif to specific
plasma membrane receptors known as integrins and are thus
indirectly associated with the intracellular actin cytoskeleton
(Hynes 1987, Burridge et al. 1988). Numerous cross-reactive
polypeptides with antibodies against extracellular matrix proteins and integrins have been reported in plant cells (Takagi et
Corresponding author: E-mail, [email protected]; Fax, +81-6-6850-5817.
This paper is dedicated to the late Dr. Eiji Kamitsubo, an outstanding light microscopist, who had made an enormous contribution to plant cell
biology.
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Cell wall–plasma membrane and mechano-perception
al. 2001). Moreover, the importance in plants of adhesions of
the plasma membrane to the cell wall has been increasingly
recognized (Wyatt and Carpita 1993, Reuzeau and Pont-Lezica
1995, Takagi et al. 2001, and references therein); for example,
in cell division (Cleary 2001), plant defense responses
(Mellersh and Heath 2001), and stability of the actin cytoskeleton (Masuda et al. 1991, Ryu et al. 1997).
Mesophyll cells of Vallisneria gigantea, an aquatic
angiosperm, exhibit actin-based cytoplasmic streaming (Liebe
and Menzel 1995, Takagi 1997). The endoplasm streams unidirectionally and rotationally along the ectoplasm that faces the
four anticlinal walls of the cell, namely, the two longer side
walls and the two shorter end walls. We have been investigating
the mechanism that maintains the organization of the bundles
of actin filaments that serve as the tracks for cytoplasmic
streaming (Takagi et al. 2001). The bundles of actin filaments
are stabilized at the end walls by a physical association of the
plasma membrane with the cell wall (Masuda et al. 1991,
Masuda et al. 1992, Ryu et al. 1995). We have shown that
RGD-sensitive and protease-sensitive factors are involved in
the mechanism (Masuda et al. 1991, Ryu et al. 1997). AntiRYD (an equivalent motif to the RGD motif) peptide antibodies cross-react with polypeptides of 54 and 27 kDa localized at
the cell wall (Ryu et al. 1997), and, moreover, anti-integrin a3
antibodies cross-react with a polypeptide of 72 kDa in the purified plasma membrane fraction (Takagi et al. 2001). On the
other hand, induction and cessation of the cytoplasmic streaming are strictly controlled by [Ca2+]cyt (Takagi and Nagai 1986).
In experiments on internodal cells of a characean alga
Chara corallina, Wayne et al. (1992) used a synthetic RGD
peptide and suggested that the plasma membrane–extracellular
matrix junction at the cell ends may play an essential role in
detecting gravitational and unidirectionally applied hydrostatic
pressure. In internodal cells of another aquatic characean alga
Nitella flexilis (Tazawa et al. 1995) and a brackish water characean alga Lamprothamnium succinctum (Okazaki et al. 1987,
Okazaki et al. 2002), hypotonic treatment induces a transient
inhibition of cytoplasmic streaming that is caused by an
increase in [Ca2+]cyt. Intracellular Ca2+ sources are involved in
the former response, whereas extracellular Ca2+ is involved in
the latter. However, it has been difficult to analyze such phenomena at the subcellular level in higher plants composed of
complex tissues. In this study, we found that cytoplasmic
streaming in the mesophyll cells of V. gigantea was rapidly and
transiently inhibited upon hypertonic treatment with sorbitol.
After precise characterization of the response using video
microscopy, we concluded that the response was a Ca2+dependent process and could be considered a mechano-sensitive process. We further examined the possible involvement of
the cell wall–plasma membrane adhesion in the mechanoperception step.
Results
A transient cessation of cytoplasmic streaming induced by
hypertonic treatment
When a mesophyll cell was treated with hypertonic artificial pond water (APW) that contained 0.4 M sorbitol, cytoplasmic streaming, the velocity of which was about 15 mm s–1, transiently ceased about 180 s after the start of treatment (Fig. 1).
A relatively slow recovery of the cytoplasmic streaming followed, taking about 420 s to reach the initial value of the velocity (Fig. 1E). Although the cell was plasmolyzed and substantially deformed several minutes after the transient cessation of
the cytoplasmic streaming (Fig. 1D, D¢), there was no substantial change in the pattern of tracks for cytoplasmic streaming
before (Fig. 1A¢) and after (Fig. 1D¢) plasmolysis. A transient
cessation of cytoplasmic streaming induced by hypertonic
treatment was always observed before plasmolysis of the cell,
and none of the cells responded to hypertonic treatment after
plasmolysis.
A transient cessation of cytoplasmic streaming was rarely
observed after treatment with sorbitol at concentrations lower
than 0.2 M but was clearly observed after treatment with sorbitol at concentrations higher than 0.3 M (Fig. 2A). Half of the
cells responded to hypertonic treatment with 0.3 M sorbitol.
The number of cells that responded to hypertonic treatment
with 0.5 M and 0.6 M sorbitol, about 75% of the cells, did not
differ significantly. At all concentrations of sorbitol examined,
a transient cessation of cytoplasmic streaming was observed
within about several seconds to 7 min after the start of hypertonic treatment. As the concentration of sorbitol became higher,
more cells responded to hypertonic treatment, and they
responded more rapidly (Fig. 2B).
Involvement of Ca2+ influx in the transient cessation of cytoplasmic streaming that was induced by hypertonic treatment
In the presence of external Ca2+ at 0.5–1.9 mM, hypertonic treatment with 0.5 M sorbitol induced a response in 50–
65% of the cells (Fig. 3A). However, a further reduction in
external Ca2+ produced a significant suppression of the response
in a concentration-dependent manner. In the absence of exterFig. 1 Effects of hypertonic treatment on the pattern and velocity of
cytoplasmic streaming on the end wall of a mesophyll cell of V.
gigantea. Optical images of the cytoplasmic layer along the end wall
of a mesophyll cell were sequentially captured before hypertonic treatment with APW containing 0.4 M sorbitol (A), immediately after
hypertonic treatment (B), at the time of transient cessation of cytoplasmic streaming (C), and several minutes after plasmolysis (D). The time
after the start of hypertonic treatment is given in seconds in the upper
right corner of each photograph. The patterns of cytoplasmic streaming
were traced and are shown by arrows in A¢–D¢ together with the outlines of cell wall and plasma membrane. The velocity of cytoplasmic
streaming was determined from the recorded images for five cytoplasmic vesicles and plotted as the mean ± SE (E). Hypertonic treatment
was started at time zero (arrowhead). N, nucleus; P, plastid. Bars =
10 mm.
Cell wall–plasma membrane and mechano-perception
1029
Fig. 2 Dependence on the concentration of sorbitol of the transient
cessation of cytoplasmic streaming in mesophyll cells of V. gigantea.
After pretreatment with a solution that contained 1 mM CaCl2 for 1 h,
specimens were treated with the solution supplemented with various
concentrations of sorbitol. The ratio of the number of cells that exhibited a transient cessation of cytoplasmic streaming (Nc) to the total
number of cells observed (Ntotal) was plotted against the concentration of sorbitol as a percentage (A). At each concentration of sorbitol,
the frequency of Nc was plotted against the duration of time between
the start of hypertonic treatment and the transient cessation of cytoplasmic streaming (B). Ntotal = 42 to 345.
Fig. 1
nal Ca2+, no cells responded to hypertonic treatment (Fig. 3A).
After treatment with ethylene glycol bis(b-aminoethylether)N,N,N¢,N¢-tetraacetic acid (EGTA) solution for 1 h, specimens
were incubated in a solution that contained 1 mM CaCl2 for
1 h. As shown in Fig. 3B, the transient cessation of cytoplas-
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Cell wall–plasma membrane and mechano-perception
Fig. 4 Effects of Ca2+-channel blockers on the transient cessation of
cytoplasmic streaming induced by hypertonic treatment in mesophyll
cells of V. gigantea. After pretreatment with a solution that contained
1 mM CaCl2 and each Ca2+-channel blocker at various concentrations
for 1 h, specimens were treated with the solution supplemented with
0.5 M sorbitol. The ratio of Nc to Ntotal (52 to 345) was plotted
against the concentration of blocker as a percentage. Solutions used in
nifedipine experiments (triangles) contained 0.5% ethanol.
Fig. 3 Effects of external Ca2+ on the transient cessation of cytoplasmic streaming induced by hypertonic treatment in mesophyll cells of V.
gigantea. After pretreatment for 1 h with a solution that contained free
Ca2+ at various concentrations adjusted using 0.1 mM EGTA, specimens were treated with the solution supplemented with 0.5 M sorbitol. The ratio of Nc to Ntotal (50 to 345) was plotted against the
concentration of Ca2+ as a percentage (A). After incubation in a solution of 0.1 mM EGTA for 1 h, specimens were treated with the solution supplemented with 0.5 M sorbitol (EGTA in B). After incubation
in a solution of 0.1 mM EGTA for 1 h followed by a solution that contained 1 mM CaCl2 for another 1 h, specimens were treated with the
Ca2+-containing solution supplemented with 0.5 M sorbitol (EGTA ®
CaCl2 in B). After incubation in a solution that contained 1 mM CaCl2
for 1 h, specimens were treated with the solution supplemented with
0.5 M sorbitol (CaCl2 in B). Ntotal = 52 to 82.
mic streaming induced by subsequent hypertonic treatment was
observed in 56% of the cells. The responsiveness was similar to
that of cells after a simple treatment with a solution that contained 1 mM CaCl2 for 1 h, without pretreatment with EGTA
solution (Fig. 3B). These experiments indicate that the response
to hypertonic treatment is not irreversibly impaired by the
EGTA solution.
Even in the presence of 1 mM CaCl2, Ca2+-channel blockers inhibited the response to hypertonic treatment in a dose-
dependent manner (Fig. 4). La3+ inhibited the response in 80%
of the cells at 0.01 mM, and completely at 0.1 mM. Although
the inhibitory effect of Gd3+, an inhibitor of stretch-activated
Ca2+ channels (Yang and Sachs 1989), seemed to be a little bit
smaller than that of La3+, Gd3+ also completely inhibited the
response at 0.1 mM. A smaller but significant inhibition of the
response was observed after treatment with nifedipine, a dihydropyridine Ca2+-channel blocker (Fleckenstein 1977), and the
response was reduced to 25% of the cells at 0.1 mM. These
results strongly suggest that hypertonic treatment induces a
Ca2+ influx across the plasma membrane through stretchactivated Ca2+ channels and that the Ca2+ in the cell induces a
transient cessation of cytoplasmic streaming.
Recovery of the response and concave-type plasmolysis induced
by hypertonic treatment
We noticed that, after a transient cessation of cytoplasmic
streaming, almost all the mesophyll cells that responded first
underwent concave-type plasmolysis in a hypertonic solution
(Fig. 5B, B¢). Hechtian strand-like structures (Reuzeau and
Pont-Lezica 1995) connected the protoplasm to the cell wall in
those cells. With further shrinkage of the protoplasm, the Hechtian strand-like structures were retracted and the protoplasm
was completely detached from the cell wall to eventually produce convex-type plasmolysis. After plasmolysis caused by the
first hypertonic treatment, the cells were deplasmolyzed in
APW lacking sorbitol (Fig. 5C, C¢). Immediately after deplasmolysis, the cells never responded to the second hypertonic
treatment with a cessation of cytoplasmic streaming. In those
cells, the plasma membrane was smoothly and completely
detached from the cell wall to instantaneously produce convextype plasmolysis (Fig. 5D, D¢). Assuming that the adhesion
Cell wall–plasma membrane and mechano-perception
1031
Fig. 6 Time course of the recovery of responsiveness to hypertonic
treatment after deplasmolysis in mesophyll cells of V. gigantea. After
plasmolysis caused by the first hypertonic treatment with 0.5 M sorbitol for 5 min, the cells were instantaneously deplasmolyzed by removing the sorbitol. During incubation after deplasmolysis, a second
hypertonic treatment with 0.6 M sorbitol was applied. The ratio of Nc
to Ntotal (41 to 83) was plotted against the time of incubation after
deplasmolysis as a percentage.
Fig. 5 Concave- and convex-type plasmolysis induced by hypertonic
treatment on the end wall of a mesophyll cell of V. gigantea. Optical
images of the cytoplasmic layer along the end wall were captured
before hypertonic treatment (A), at the time of concave-type plasmolysis induced in APW containing 0.5 M sorbitol (B), immediately after
deplasmolysis in fresh APW (C), and at the time of convex-type plasmolysis induced by the second hypertonic treatment with APW containing 0.6 M sorbitol (D). The outlines of cell wall and plasma
membrane were traced and are illustrated in A¢–D¢. N, nucleus; P, plastid. Bars = 10 mm.
between the cell wall and the plasma membrane plays some
role in the transient cessation of cytoplasmic streaming induced
by hypertonic treatment, we examined the recovery of the
response to a second hypertonic treatment after deplasmolysis
(Fig. 6), together with observations of the type of plasmolysis.
Although the cells hardly responded to a second hypertonic
treatment after 6 h of deplasmolysis, responsiveness was gradually recovered in about 15%, 35%, and 60% of the cells after
12, 18, and 24 h of deplasmolysis, respectively. After 24 h of
deplasmolysis, the cells again exhibited concave-type plasmolysis, as shown in Fig. 5B and B¢, concomitantly with the recovery of responsiveness to the second hypertonic treatment.
Involvement of protein synthesis in the recovery of the response
to hypertonic treatment
We asked whether protein synthesis is necessary for the
recovery of responsiveness to hypertonic treatment. In the presence of 10 mM cycloheximide, a general protein synthesis
inhibitor, the responsiveness to hypertonic treatment was not
recovered at all, even after 24 h of deplasmolysis (Experiment
1 in Table 1). An RNA synthesis inhibitor cordycepin also
inhibited the recovery of the response to about 13% of the cells
at 10 mM (Experiment 1 in Table 1). However, after a simple
treatment with 10 mM cycloheximide or 10 mM cordycepin for
24 h, the cells responded normally to the first hypertonic treatment. On the other hand, after a simple treatment with 1 mM
cycloheximide for 24 h, the cells did not respond even to the
first hypertonic treatment (data not shown).
Next, we examined the effects of an exogenously applied
protease on the recovery of responsiveness. After 24 h of
deplasmolysis in the presence of 110 units mg–1 of trypsin at
500 mg ml–1, the response to a second hypertonic treatment was
reduced to 24% (Experiment 1 in Table 1). However, after a
simple treatment with 500 mg ml–1 trypsin for 24 h, the cells
responded to the first hypertonic treatment almost normally. On
the other hand, after 24 h of deplasmolysis in the presence of
boiled trypsin at 500 mg ml–1, the cells responded normally to a
second hypertonic treatment (data not shown). These results
suggest that protein synthesis is necessary for restoration of the
responsiveness to hypertonic treatment and that protease-sensitive factors are involved in the mechanism. Moreover, in the
presence of each of those three different kinds of reagent, we
confirmed that most of the non-responded cells exhibited convex-type plasmolysis, whereas the cells retrieved the respon-
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Cell wall–plasma membrane and mechano-perception
Table 1 Effects of cycloheximide, cordycepin, trypsin, and an RGD peptide on the responsiveness to hypertonic treatment in mesophyll cells of V. gigantea
First treatment
Experiment 1
Cycloheximide
Cordycepin
Trypsin
Control
Experiment 2
RGD peptide
RGE peptide
Control
Second treatment
Nc/Ntotal (%)
Ntotal
Nc/Ntotal (%)
Ntotal
55.9
72.1
68.1
73.4
68
64
72
64
0
12.9
24.3
63.5
65
62
70
115
30.2
30.4
37.1
53
46
35
In Experiment 1, the first hypertonic treatment with 0.6 M sorbitol was applied to cells after incubation for
24 h in APW containing either cycloheximide at 10 mM, cordycepin at 10 mM, or 110 U mg–1 of trypsin at
500 mg ml–1 (First treatment). The second hypertonic treatment was applied in a similar incubation regime
after cells were plasmolyzed and subsequently deplasmolyzed as described in the legend to Fig. 6 (Second
treatment). In Experiment 2, the second hypertonic treatment was applied after incubation for 24 h either
with an RGD peptide at 30 mM or with an RGE peptide (a control peptide) at 30 mM following plasmolysis
and deplasmolysis. Control; incubation in normal APW (Experiment 1) or in a buffer solution that did not
contain either peptide (Experiment 2).
siveness to second hypertonic treatment exhibited concave-type
plasmolysis (data not shown).
Effects of an RGD peptide on the response induced by hypertonic treatment
We treated the cells with an RGD peptide to examine
whether an RGD-sensitive adhesion of the plasma membrane to
the cell wall is responsible for the response to hypertonic treatment. After incubation in the presence of the RGD peptide for
24 h, mesophyll cells exhibited an abnormal pattern of tracks
for cytoplasmic streaming on the end wall (Fig. 7A¢–D¢); however, these cells responded almost normally to hypertonic treatment (Fig. 7E).
Next, we examined the effects of the RGD peptide on the
recovery of responsiveness to hypertonic treatment. After 24 h
of deplasmolysis in a solution containing 30 mM of an RGD
peptide, about 30% of cells responded to a second hypertonic
treatment (Experiment 2 in Table 1). In the presence of 30 mM
of an RGE (Arg-Gly-Glu) peptide, used as a control peptide,
about 30% of cells also responded to a second hypertonic treatment. Moreover, after 24 h of deplasmolysis in a buffer solution that did not contain either peptide, a response to the second
hypertonic treatment was induced in about 37% of the cells.
These results indicate that an RGD-sensitive adhesion is
involved in the organization of the actin cytoskeleton, but it is
probably not involved in the response to hypertonic treatment.
Discussion
Transient cessation of cytoplasmic streaming induced by hypertonic treatment
In this study, we found that cytoplasmic streaming in mesophyll cells of V. gigantea transiently ceased several seconds to
several minutes after hypertonic treatment with sorbitol. The
response was essentially an all-or-none response, and it was
transient even when the hypertonic treatment was prolonged
(Fig. 1E), indicating that receptor systems and/or subsequent
steps in the signal transduction pathway show adaptive characteristics. The cells hardly responded to hypertonic treatment
with sorbitol at concentrations lower than 0.2 M (Fig. 2A),
indicating that perhaps this concentration was close to a
response threshold. In a parallel experiment, we observed that
mesophyll cells that exhibited a sign of plasmolysis first
appeared at 0.2 M sorbitol. As the concentration of sorbitol
increased, the response was induced more rapidly and in more
cells (Fig. 2B). This dose dependency indicates that the concentration of sorbitol might correspond to the intensity of stimulus.
The rapid and transient cessation of cytoplasmic streaming
induced by hypertonic treatment reminds us of transient phenomena reported in plant cells induced by various mechanical
stimuli. Knight et al. (Knight et al. 1991, Knight et al. 1992)
reported that a transient increase in [Ca2+]cyt was induced
immediately after touch or wind stimuli in tobacco plants
genetically transformed with apo-aequorin. When wind stimuli
were quantitatively applied, the response intensity was dose
dependent (Knight et al. 1992). Several electrophysiological
studies on the perception of mechanical stimuli have also been
Cell wall–plasma membrane and mechano-perception
carried out in internodal cells of Characeae (Staves and Wayne
1993, Shimmen 1996). Shimmen (1996) developed an efficient
system to apply mechanical stimuli quantitatively to a single
internodal cell of Chara corallina: the cell was stimulated by
1033
dropping a glass tube onto it, and both the weight of the tube
and the height from which it was dropped could be varied. In
the internodal cells of this species, mechanical stimuli larger
than a threshold value induce the generation of an action potential followed by a rapid cessation of cytoplasmic streaming. In
the present material, we confirmed that touch also induces a
transient cessation of cytoplasmic streaming within a few seconds (data not shown).
Although possible perception mechanisms for touch or
hypotonic treatment have been proposed in charophyte cells,
those for hypertonic treatment are unknown (Shepherd et al.
2002). As discussed below, when the adhesion between the cell
wall and the plasma membrane is maintained to be sufficiently
strong, the cell wall may not allow the protoplasm to decrease
its surface area upon hypertonic treatment. This nature of the
adhesion might instantaneously produce a stretching force to
the plasma membrane, which could activate mechano-sensitive
ion channels. The complete inhibition by Gd3+ of the transient
cessation of cytoplasmic streaming induced by hypertonic
treatment (Fig. 4) strongly suggests the involvement of stretchactivated Ca2+ channels in the present response. Therefore,
since the response to hypertonic treatment was rapidly induced
in a dose-dependent manner and was sensitive to Gd3+, we conclude that hypertonic treatment is one mechanical stimulus that
acts on V. gigantea mesophyll cells. We have thus succeeded in
precisely analyzing intracellular events associated with the perception of mechanical stimuli in a multicellular plant by realtime observations of the transient cessation of cytoplasmic
streaming.
Involvement of Ca2+ in the response to hypertonic treatment
The response to hypertonic treatment depended strictly on
the presence of extracellular Ca2+ (Fig. 3) and was completely
inhibited, even in the presence of Ca2+, after treatment of cells
with Ca2+-channel blockers (Fig. 4). These results indicate that
an influx of extracellular Ca2+ plays an essential role in the
response to hypertonic treatment. A Ca2+-sensitive motor activity, which can translocate filamentous actin in vitro in the presence of ATP, has been partially fractionated from V. gigantea
leaves (Takagi et al. 1995). This motor activity might mediate
Fig. 7 Effects of RGD peptide on the pattern of cytoplasmic streaming and the response to hypertonic treatment on the end wall of a mesophyll cell of V. gigantea. After incubation in a solution that contained
3 mM RGD peptide for 24 h, optical images of the cytoplasmic layer
along the end wall were captured before hypertonic treatment with
APW containing 0.4 M sorbitol (A), immediately after hypertonic
treatment (B), at the time of transient cessation of cytoplasmic streaming (C), and several minutes after plasmolysis (D). The time after the
start of hypertonic treatment is given in seconds in the upper right corner of each photograph. The patterns of cytoplasmic streaming were
traced and are shown by arrows in A¢–D¢ together with the outlines of
cell wall and plasma membrane. From the recorded images, the velocity of cytoplasmic streaming was determined as described in the legend to Fig. 1 (E). Hypertonic treatment was started at time zero
(arrowhead). P, plastid. Bars = 10 mm.
1034
Cell wall–plasma membrane and mechano-perception
between the increase in [Ca2+]cyt caused by the influx of extracellular Ca2+ and the cessation of cytoplasmic streaming. The
hypertonic treatment-induced cessation of cytoplasmic streaming was suppressed completely by La3+ and significantly by
nifedipine (Fig. 4) but was insensitive to the phenylalkylamine
blocker verapamil (data not shown). Takagi and Nagai (Takagi
and Nagai 1985, Takagi and Nagai 1988) reported that the farred light-induced cessation of cytoplasmic streaming in V.
gigantea was also suppressed completely by La3+ and partially
by nifedipine, but not at all by verapamil. A similar sensitivity
to different kinds of Ca2+-channel blockers may indicate that
mechanical stimuli and light share, at least in part, the signaling
pathways leading to the cessation of cytoplasmic streaming. In
the case of the fern Adiantum capillus-veneris, Sato et al.
(2001) also showed that an influx of external Ca2+, which they
inhibited with La3+ and Gd3+, is important for the early signaling steps in chloroplast mechano-relocation. However, these
Ca2+-channel blockers had no effect on chloroplast photorelocation induced by red and blue light.
While Ca2+ regulates many important cellular functions,
including ionic balance, motility, gene expression, mitosis, and
secretion (Bush 1995, and references therein), physiological
roles of the Ca2+-dependent cessation of cytoplasmic streaming
induced by hypertonic treatment are unclear. Ca2+ itself should
act as a second messenger in the transduction pathways of several cellular processes. Alternatively, it might be general in
plants that cytoplasmic motility is transiently reduced by
mechanical stimuli (Okazaki and Tazawa 1986, Staves and
Wayne 1993). Such a transient reduction in cytoplasmic motility might also act as an important message affecting downstream responses. Several pharmacological studies have demonstrated that not only extracellular Ca2+ (Haley et al. 1995,
Knight et al. 1997, Takahashi et al. 1997) but also intracellular
Ca2+ (Knight et al. 1992, Haley et al. 1995, Knight et al. 1997)
is involved in a transient increase in [Ca2+]cyt induced by
mechanical stimuli. Although a possible involvement of intracellular Ca2+ sources in the present response has not yet been
investigated, plant cells seem to respond to mechanical stimuli
using at least two different mechanisms, a Ca2+ influx across
the plasma membrane and a Ca2+ release from intracellular
sources, to generate mechano-sensory Ca2+ signals.
Roles of the cell wall–plasma membrane adhesion in the
response to hypertonic treatment
Although the cells seemed to return almost to their original shape immediately after plasmolysis and subsequent
deplasmolysis (Fig. 5C, C¢), the deplasmolyzed cells never
responded to a second hypertonic treatment administered right
away (Fig. 6). Responsiveness was gradually recovered during
24 h of incubation. These results suggest that plasmolysis disrupts the plausible mechanism of the response by physically
detaching the plasma membrane from the cell wall, and that
simple reattachment of the plasma membrane to the cell wall is
insufficient to functionally recover the mechanism. In this
study, we found that while responsive cells to hypertonic treatment almost always exhibited concave-type plasmolysis, as
shown in Fig. 5B and 5B¢, non-responsive cells exhibited convex-type plasmolysis, as shown in Fig. 5D and 5D¢. The type of
plasmolysis observed in a hypertonic solution may reflect the
nature of adhesion between the cell wall and the plasma membrane, such as the mode and/or strength of adhesion: for example, concave-type plasmolysis is induced when the adhesion is
strong, whereas convex-type plasmolysis is induced when it is
weak (Oparka 1994). When the adhesion is strong, shrinkage
of the protoplasm induced by hypertonic treatment may apply a
stretching force to the plasma membrane prior to its detachment from the cell wall, whereas this may not occur when the
adhesion is weak. Consequently, we propose that intact adhesion of the plasma membrane to the cell wall is essential for the
response to hypertonic treatment and is probably responsible
for the opening of stretch-activated Ca2+ channels.
The inhibitory effects of EGTA on the response (Fig. 3)
might be attributable not only to depletion of extracellular Ca2+
but also to the loss of responsiveness due to changes in the
nature of adhesion between the cell wall and the plasma membrane. Nevertheless, the crucial requirement of extracellular
Ca2+ to the response is evident because Ca2+ channel blockers
completely inhibited the response even in the presence of extracellular Ca2+ (Fig. 4).
Pickard and Ding (1993) proposed an attractive model
involving a “plasmalemmal control centre”, which contains
several regulatory proteins, including mechano-sensory Ca2+
channels. In their model, Pickard and Ding presume that the
mechanical stimuli that serve as the primary signal for the
mechano-sensory Ca2+ channels must be transmitted to the
plasma membrane by a specific wall-to-membrane linker, analogous to the integrin family of transmembrane proteins.
Although molecules responsible for the adhesion remain
unknown, Wyatt and Carpita (1993) reported evidence for a
cell wall–plasma membrane–cytoskeleton continuum that can
be interfered with by an RGD peptide. In the present study, we
demonstrated that protein factor(s), whose function is impaired
after plasmolysis, might be involved in the response to hypertonic treatment (Experiment 1 in Table 1). Substantial suppression of the recovery of the responsiveness by trypsin strongly
suggested that at least a part of relevant factor(s) is located on
the cell surface. Since the anti-RYD peptide (Ryu et al. 1997)
and anti-integrin a3 (Takagi et al. 2001) antibodies detected
specific polypeptides in the present material, we have started to
examine a possible involvement of such factors in the response
using the RGD peptide.
Although the pattern of cytoplasmic streaming was sensitive to the RGD peptide (Fig. 7), as already reported by Ryu et
al. (1997), the response to hypertonic treatment was insensitive
(Fig. 7E). Moreover, neither the RGD peptide nor the RGE
peptide had a significant effect on the recovery of responsiveness after plasmolysis and deplasmolysis (Experiment 2 in
Table 1). From these results, we suppose that an RGD-sensitive
Cell wall–plasma membrane and mechano-perception
adhesion of the plasma membrane to the cell wall is probably
not involved in the sensing of mechanical stimuli caused by
hypertonic treatment. However, we cannot totally exclude an
involvement of an integrin in the mechanism, since the same
integrin molecule could bind not only to RGD-containing proteins but also to proteins with other motifs (Hynes 1992). On
the other hand, a Ca2+ channel is also a possible candidate for
one of the protein factors whose function is impaired after plasmolysis. Intact Ca2+-channel proteins and/or the normal distribution of Ca2+ channels might play an important role in the perception of mechanical stimuli. These possibilities remain to be
investigated.
Materials and Methods
Plant material and pretreatment of specimens
Vallisneria gigantea Graebner was cultured under a cycle of 12 h
of darkness and 12 h of light (0.5 W m–2 from white fluorescent lamps)
at 20–25°C, as described by Izutani et al. (1990). The procedures for
pretreatment of specimens for experiments have been described in
detail elsewhere (Ryu et al. 1995). Briefly, a leaf segment was cut into
small pieces about 0.8 mm long along the transverse axis of the leaf to
expose the end walls of the cells. The trimmed pieces of leaves were
incubated in a plastic vessel with 50 ml of APW (0.05 mM KCl,
0.2 mM NaCl, 0.1 mM Ca(NO3)2, 0.1 mM Mg(NO3)2, and 2 mM piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) buffer at pH 7.0) under
the original light regimen for 36–48 h.
Microscopic observation
Each specimen was fastened onto a slide glass using a small
amount of Vaseline so as to expose the end walls of mesophyll cells,
and was covered with a drop of the incubation solution. For hypertonic treatment, the specimen was perfused with the solution supplemented with various concentrations of sorbitol under video microscopy. The specimen was continuously observed under an inverted light
microscope (IMT-2; Olympus, Tokyo, Japan) with a 40´ objective lens
(NA 0.55). Optical images of the cytoplasmic layer along the end walls
of mesophyll cells were recorded with a television camera (WV-1550;
National, Kadoma, Japan) and stored on videotape with a video
recorder (NV-FS5; National, Kadoma, Japan). A green interference filter (G530; Olympus, Tokyo, Japan) was used for observation to avoid
any light damage. On the recorded optical images, the five most rapidly moving cytoplasmic vesicles were chosen, and the velocity of
cytoplasmic streaming was calculated from the distance that each cytoplasmic vesicle moved in 1 s.
When the mesophyll cells in a larger number of specimens were
plasmolyzed and deplasmolyzed, specimens were first incubated in a
glass vessel with 4 ml of APW supplemented with 0.5 M sorbitol for
5 min. We confirmed by video microscopy that almost all mesophyll
cells in the incubated specimens exhibited plasmolysis. Then, the concentration of sorbitol was decreased gradually at a rate of about
100 mM min–1 to avoid any possible damage that a sudden increase in
the turgor pressure might cause.
Solutions
Solutions of various concentrations of Ca2+ were prepared by
using an EGTA solution, which contained 0.1 mM EGTA and 30 mM
PIPES buffer at pH 7.0, supplemented with various concentrations of
CaCl2. The concentration of free Ca2+ was calculated using the association constant between EGTA and Ca2+ (Fabiato and Fabiato 1978).
1035
LaCl3 and GdCl3 were dissolved in distilled water as stock solutions of
10 mM. Nifedipine (Nacalai Tesque, Kyoto, Japan) was dissolved in
ethanol as a stock solution of 20 mM. Each stock solution was diluted
with a buffered solution that contained 1 mM CaCl2 and 30 mM PIPES
buffer at pH 7.0 to an appropriate concentration at the time of use. Pretreated specimens were incubated in the solution for 1 h before hypertonic treatment.
Cycloheximide, cordycepin (Sigma Chemical Co., St. Louis,
MO, U.S.A.), and trypsin (Boehringer Mannheim Gmbh, Mannheim,
Germany) were dissolved directly in APW at the concentration indicated in the text. The synthetic hexapeptides GRGDSP (RGD peptide)
and GRGESP (RGE peptide) were purchased from Asahi Techno
Glass Co. (Chiba, Japan) and then dissolved in a buffered solution that
contained 0.1 mM KCl, 0.1 mM NaCl, 1 mM EGTA, 10 mM free Ca2+
ions (added as CaCl2), and 10 mM HEPES, with the pH adjusted to 7.0
with NaOH, as described by Ryu et al. (1997). After pretreatment with
the solution at the concentration and for the period of time indicated in
the text, specimens were incubated in a buffered solution that contained 1 mM CaCl2 and 30 mM PIPES buffer at pH 7.0 for 1 h before
hypertonic treatment.
Acknowledgments
This work was partly supported by the 21st Century COE program and a Grant-in-Aid for Scientific Research No. 08640827 from
the Ministry of Education, Culture, Sports, Science and Technology of
Japan.
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(Received January 21, 2003; Accepted July 22, 2003)